KR102108876B1 - Methods and compositions for stabilizing dried biological materials - Google Patents

Abstract

The present invention relates to a method for preparing a dried formulation of a biopharmaceutical with the aim of minimizing the loss of activity of the biopharmaceutical upon drying and providing a dried formulation with an extended shelf life. The method includes drying an aqueous solution comprising at least one amino acid, polyol and metal salt, in addition to the biopharmaceutical. Preferably, the amino acid is glutamate, the polyol is sorbitol, and also optionally mannitol, and the metal salt is a magnesium salt. The solution is dried by vacuum drying or freeze drying. The method is particularly useful for preparing dried formulations of viruses such as poliovirus or respiratory syncytial virus used for vaccines. The invention also relates to dried formulations prepared according to the methods of the invention and their use as medicaments, for example vaccines.

Description

METHODS AND COMPOSITIONS FOR STABILIZING DRIED BIOLOGICAL MATERIALS

The present invention relates to methods and compositions for stabilizing and preserving dried biological material. Such biological materials include materials for the preparation of lyophilized or vacuum-dried biologics, such as vaccines, especially viral vaccines. Accordingly, the present invention relates to the field of manufacture and formulation of biopharmaceuticals and to the field of vaccines.

Given the fact that biological compounds are usually fragile and vulnerable, long-term storage of biological compounds poses special challenges. In a liquid environment, little is sufficiently stable in solution or suspension, so that biological compounds can be stored in solution for longer than a short period of time in isolation, purification and non-refrigerated conditions, especially at room temperature or higher.

One way to improve the stability of biopharmaceuticals is to convert them into a dry state [17]. Commercially and practically, storage as a biologic in dry form has enormous advantages. Successfully dried formulations, materials, and tissues have reduced weight and require reduced storage space despite their increased shelf life. It is intended to stockseedlots, bulk, or final lots, as well as use for the final product, such as in a final high dose vaccine for use within 3 months to 2 years. Storage of the dried material at room temperature is much more cost effective than the low temperature storage option, and the accompanying costs are therefore much more cost effective. In addition, several delivery routes, including pulmonary delivery of powders, skin delivery by coated or dissolved microneedle, and parenteral delivery by powders or soluble needles, depend on the precise manner of the formulation. Several techniques exist for making dried biological compounds, including spray drying, vacuum drying, air-drying coatings, and foam-drying. One of the oldest and most commonly used techniques is freeze-drying, also called freeze-drying. Freeze-drying for a long time has been seen as a technology rather than a science, which hinders scientific approaches and research.

The most commonly used method for preparing solid biologics is freeze-drying. This process consists of two drying steps followed by a freezing step, in which the frozen water is removed by sublimation, and the second drying is the non-frozen 'bound' water. . Freeze or dry stress can alter the thermodynamic stability of a biopharmaceutical and can induce or promote protein unfolding. Unfolding can cause irreversible denaturation of biologics, but can also decrease storage stability in dry conditions [19]. For stable lyophilisates, excipients provided as stabilizers and / or extenders are used. Other compounds such as sugars, polymers, amino acids and surfactants have been shown to improve the stability of biopharmaceuticals during lyophilization and subsequent storage [18, 20]. In the literature, it is described how excipients are believed to protect biologics such as proteins and vaccines during freeze, drying and subsequent storage. Understanding the freezing and cryoprotection mechanisms of various stabilizers is important for the development of rational formulations and process designs for stable lyophilized vaccines [21].

During freezing, the physical environment of the biopharmaceutical dramatically changes what causes the development of stress that affects the integrity of the proteinaceous biopharmaceutical. The most critical stresses for biopharmaceuticals exposed during freezing are low temperature, freeze concentration and ice formation [18, 19, 21, 22]. Cold denaturation is a phenomenon in which biologics lose their compact folded structure as a result of temperature degradation. The currently accepted explanation for cold denaturation is based on changes in the contact free energy between water and non-polar groups at colder temperatures, which weakens the hydrophobic interaction and thus destroys the biopharmaceutical structure [19, 20, 23]. ]. Due to ice formation, the concentration of all solutes increases dramatically during freezing. Any changes related to concentration such as ionic strength, solute crystallization and phase separation can potentially destabilize biologics [21].

The initial relative composition and pH of the formulation can avoid deterioration of the biologic due to freezing stress. For example, increasing the biopharmaceutical concentration in a formulation relative to other excipients prior to freezing has been found in many biopharmaceuticals to increase the stability of the biopharmaceutical during freeze-thaw [24]. Likewise, an initial pH that is optimal for a biopharmaceutical in solution will provide maximum recovery of the original biopharmaceutical after freeze-thaw [20].

Even after optimization of all these factors, many biologics still denature after freeze-thaw. Therefore, additives are required to minimize protein / biopharmaceutical denaturation. Various excipients from very different chemical classes can provide cryoprotection. According to the 'solute exclusion hypothesis', cryoprotectants did not appear to exist preferentially in contact with the surface of biologics in aqueous solutions [24]. The thermodynamic phenomenon of solute exclusion in the presence of various biologics has been investigated for various excipients such as salts, amino acids, methylamines, polyethylene glycols, polyols, surfactants and sugars [19, 21, 24, 25].

The 'vitrification hypothesis' is a well-known kinematic mechanism. According to this mechanism, both freeze concentration and temperature drop increase viscosity, reduce mobility and slow all kinetic processes. When the system reaches the vitreous state, all molecules in the glass are physically (eg, denatured, aggregated) and chemically fixed (eg, oxidation, hydrolysis, deamidation), and the rate constant of biopharmaceutical degradation is reduced [ 19, 26].

The ice-water interface formed during freezing can cause surface denaturation. The addition of surfactants can reduce the surface tension of the biopharmaceutical solution, thus reducing biopharmaceutical adsorption and aggregation [25, 27].

Polymers can stabilize biopharmaceuticals by increasing the glass transition temperature of the formulation and by inhibiting the crystallization of small stabilizing additives such as disaccharides [18, 22]. Amino acids can also reduce the rate and extent of buffer salt crystallization, thereby inhibiting pH changes, thereby protecting biologics from cryo-denaturation [18].

In aqueous solution, the biopharmaceutical is fully hydrated, which means that the biopharmaceutical has a monolayer of water that covers the biopharmaceutical surface (by hydrogen bonding) and interacts with it [28]. Drying removes a portion of the hydrated outer layer, which can disrupt the native state of the biologic and cause denaturation. A protective agent is required to suppress denaturation during drying. An important stabilization mechanism of these protective agents is called the 'water substitution hypothesis' [18-20, 29]. Sugars such as sucrose and trehalose, polyols [20, 30], and amino acids [31] can form hydrogen bonds with dried biologics. Accordingly, they can serve as water substitutes when the hydrated outer layer is removed. The formation of amorphous glass, described by the 'vitrification hypothesis' above, is also a major protective mechanism.

In addition to water substitution and glass formation, many excipients, especially polymers, stabilize biologics by increasing the glass transition temperature (Tg), defined as the transition temperature between the rubber (liquid) state and the glassy (solid) state. I can do it. In general, the higher the Tg, the lower the molecular mobility (e.g., migration of biopharmaceuticals, stabilizing compounds, oxygen and water) in glass, resulting in more stable biopharmaceutical formation during drying and subsequent storage [18, 22]. Another mechanism involved in the stabilization of biopharmaceuticals during drying and applicable to polysaccharides and other polymers is inhibition of crystallization during solute concentration of small excipients that stabilize the biopharmaceuticals during drying.

Due to their preference for the bulk environment instead of the biopharmaceutical surface (previously referred to as 'solute exclusion'), some excipients may act as bulking agents, some excipients providing mechanical support for the final cake. It provides, improves product precision, and suppresses product collapse during drying. Mannitol and glycine are often used as extenders because of their non-toxic, high solubility and high eutectic temperature [18, 25, 32]. Most amino acids are potential extenders and are also readily crystallized [33].

If the biopharmaceutical is stable during the drying process, long-term storage in dry conditions is often feasible. In general, although the drying process itself is mostly detrimental to biopharmaceuticals, dried biopharmaceuticals may still lose their structure or efficacy during storage if not properly formulated. Aggregation is the main physical instability to biologics during storage. Other chemical degradations, such as deamidation, oxidation and hydrolysis, can also occur during storage, but this change does not necessarily affect the activity of the biologic and depends on the location of the modified residue (s). Reducing sugars, such as glucose and sucrose, can react with lysine and arginine residues in biopharmaceuticals to form carbohydrate adducts through the Maillard reaction, browning reaction, which is significant for freeze-dried biopharmaceuticals during storage. It can cause loss of activity.

Storage temperature is one of the most important factors affecting the stability of the biologic in the solid state. Other factors affecting long-term storage stability are the glass transition temperature (Tg) of the formulation, the pH of the formulation and the residual moisture content after drying. The moisture content of the dried formulation can change significantly during storage due to several factors, including plug moisture release, crystallization of the amorphous excipient or moisture release from the excipient hydrate. Excipients used to stabilize the biologic during drying can render the biopharmaceutical unstable in the solid state if their amounts are not properly used in the formulation. In addition, because the crystalline state is more thermodynamically stable than the glassy state, there is a risk of crystallization of the amorphous excipient during storage [39]. Many saccharides and polyols tend to crystallize, but this is strongly influenced by their relative amounts in the formulation, storage temperature and relative humidity [18, 40]. In addition, the relative composition of various excipients and the presence of non-crystallization stabilizers such as polymers can inhibit crystallization of the excipients.

The stabilization mechanism for biopharmaceuticals in a dry state during long-term storage is similar to that for lyoprotection, including the formation of an amorphous free state, water displacement and hydrogen bonding between excipients and biopharmaceuticals [41]. The combination of these mechanisms requires maximum biopharmaceutical stabilization in the solid state [18, 42]. The final quality of the lyophilized product is determined by the selection of excipients including buffers, extenders and stabilizers and the freeze drying process.

Polio is a highly infectious disease that affects mostly young children. Diseases caused by any of the three serotypes of poliovirus (type 1, type 2 or type 3) do not have specific therapeutic agents, but can be prevented through immunization. Currently, the oral polio vaccine (Sabin OPV) is a vaccine chosen to struggle against the global eradication of polio. However, the main problem is the ability of the OPV to convert into a form that can cause paralysis, so-called vaccine-related paralysis of polio (VAPP). Another risk of permanent use of live attenuated poliovirus is the return to vaccine-derived poliovirus (VDPV) [1]. In Western countries, the use of inactivated Salk polio vaccine (IPV) is the current preferred method for eliminating the risk of VAPP and circulating VDPV. IPV is considered to be the most appropriate for the continuation of the global eradication program [1-3].

To achieve global polio eradication, (enhanced) IPV must be effective, inexpensive, safe to manufacture, and easy to administer [4]. Currently, the viability of IPV in developing countries is limited. This is because IPV is more expensive than OPV and is administered only by injection [1, 3, 5]. To limit the cost of IPV, WHO and RIVM are developing non-profit IPVs for technology transfer to developing countries. The new vaccines will be based on the OPV strain, Sabin (sIPV), because the blockade of wild-type salk polio virus during manufacture can be particularly problematic in developing countries, and it is also expected that manufacturing costs for this will be less expensive. To further reduce costs, RIVM is developing a sIPV formulation that indicates a reduction in dosage using adjuvants and / or other immunization routes [6].

Several variants of vaccine delivery methods are currently under development, as modified delivery methods and improved vaccine formulations have the potential to make vaccine delivery easier and safer [7, 8].

There is a difference in thermal stability between the various inert folio serotypes, with type 1 being the most vulnerable. In the presence of any preservative, Type 1 slowly deteriorates after storage at 4 ° C. for 2 years, while Types 2 and 3 retain efficacy for many years. The D-antigen content is markedly reduced after 20 days at 24 ° C, and detection is impossible after exposure at the same period at 32 ° C. In contrast, for Type 2, there is no significant change in D-antigenicity at this temperature. Type 3 remains stable at 24 ° C for 20 days, but the D-antigen content at 32 ° C is significantly lowered [9].

All three serotypes of IPV were incorporated into the mixed vaccine and ranged from 1 year to 4 years or more, based on observations made in the DT-folio vaccine preserved with 2-phenoxy-ethanol and adsorbed to aluminum hydroxide. When stored at 4 ° C for a period, it shows satisfactory retention [9, 10]. Longer storage resulted in a decrease in antigenicity, especially for type 1 [9]. As a standalone vaccine, IPV is stable at 4 ° C for 4 years and 25 ° C for 1 month [10]. There is a significant loss of efficacy of type 1 after 1-2 days at 37 ° C., and a significant loss of efficacy of types 2 and 3 after 2 weeks [11, 12]. In addition, freezing adversely affects the efficacy of IPV, which is associated with the loss of D-antigen structure [12].

After polio eradication, stockpiles of polio vaccine are required to predict the potential risk of new polio outbreaks caused by circulating VDPV (even after OPV discontinuation) or by terrorist attacks using biological weapons [13-15]. In order to obtain optimal vaccine stocks, various problems need to be considered. Shelf life is an important detail because extended expiration times reduce stockpiling costs [16]. To ensure the efficacy of the vaccine for many years, the shelf life of vaccines such as IPV needs to be extended. Accordingly, there is a need for improved formulations that extend the shelf life of biopharmaceuticals, and there is a need for improved methods of preparing such formulations, preferably in dry form.

In a first aspect, the present invention relates to a method for preparing a biopharmaceutical in a dried formulation, comprising drying a solution comprising a biopharmaceutical, amino acid, polyol, metal salt and water.

Preferably, in the method of the present invention, the solution is essentially 1 pg-10 g of biopharmaceutical per ml, 0.01-20% (w / v) amino acid, 0.5-20% (w / v) polyol, 0.005-10 % (w / v) metal salt and water. More preferably, the amino acid is glutamate, arginate, histidine, asparagine, glycine or mixtures thereof; Polyols are sorbitol, mannitol, mannose, maltitol or mixtures thereof; And the metal salt is Mg ⁺⁺ , Ca ⁺⁺ or Li ⁺ or mixtures thereof, where Cl ^- or SO ₄ ^{2 -} salts or mixtures thereof are preferred.

In the method of the present invention, glutamate is preferably dissolved in a solution in the form of monosodium glutamate.

In a preferred embodiment of the method of the invention, the solution is essentially 1 pg-10 g of biopharmaceutical per ml, 5-20% (w / v) sorbitol, 0.5-20% (w / v) monosodium glutamate, 2 -10% (w / v) magnesium salt, preferably MgCl ₂ and / or MgSO ₄ , and optionally 5-20% (w / v) mannitol.

In the method of the present invention, the solution preferably contains a pharmaceutically acceptable buffer, and is buffered with a neutral pH. In addition, the solution is preferably dried by vacuum drying or freeze drying.

In the method of the invention, the biopharmaceutical is preferably an agent comprising a proteinaceous substance. More preferably, the biologic is a virus, preferably Enterovirus or pneumovirus . More preferably, the biologic is a serotype 1, 2 and 3 poliovirus, preferably serotype 1, 2 and 3 inactivated poliovirus, or wherein the biologic is human or bovine RSV.

In a preferred embodiment of the method of the invention, the dried formulation retains at least 50% of the activity of the biopharmaceutical present in the solution prior to drying, upon reconstitution after drying. In another embodiment of the method of the present invention, the solution to be dried comprises one or more other biologics, wherein the difference in activity loss for the other biologics is less than 50%, and thus the organism with the highest loss of activity The retention activity of the drug is expressed as a percentage of the retention activity of the biologic with the lowest loss set to 100%.

In a preferred embodiment of the method of the invention, the dried formulation retains at least 50% of the activity of the biopharmaceutical present in the solution prior to drying, upon reconstitution after storage at 45 ° C. for at least one week. In another embodiment of the method of the present invention, the dried formulation comprises one or more other biologics, wherein the difference in activity loss for the other agent is preferably less than 50%, thus having the highest loss of activity. The retention activity of a biologic is expressed as a percentage of the retention activity of a biologic with the lowest loss set to 100%.

In a second aspect, the invention relates to a dried formulation of a biopharmaceutical obtainable by any method according to the invention.

In a third aspect, the present invention relates to a formulation according to the invention for use as a medicament, preferably to a formulation according to the invention for use as a medicament for inducing an immune response (in an individual) against an infectious disease or tumor. will be.

Figure 1 D-antigen recovery immediately after lyophilization of serotypes 1, 2 and 3 IPV-formulations (Experiment A). The stabilizing effects of sucrose (SUC), trehalose (TREH), mannitol (MAN), dextran (DEX) and sodium chloride (NaCl) were tested.
Figure 2 Response surface plot showing percent recovery of D-antigens of these 3 serotypes immediately after lyophilization of IPV-formulations containing 10% mannitol and 0% dextran.
Figure 3 D-antigen recovery immediately after lyophilization of serotypes 1, 2 and 3 IPV-formulations (Experiment B). The stabilizing effects of sucrose (SUC), trehalose (TREH), monosodium glutamate (MSG), hydroxyethyl starch (HES) and sodium chloride were tested.
Figure 4 D-antigen recovery immediately after vacuum drying (V), freeze drying using a slow freezing rate (L) and freeze drying using a fast freezing rate (S). The stabilizing effect of sucrose and trehalose compared to the IPV vaccine without negative stabilizer (negative control) was investigated.
Recovery of D-antigens immediately after lyophilization of serotypes 1, 2 and 3 IPV-formulations as shown in Figure 5 (Experiment C)
6 D-antigen recovery immediately after lyophilization of serotypes 1, 2 and 3 IPV-formulations (Experiment D). All formulations contained 5% sorbitol and 125 mM NaCl, and 5% peptone and further as indicated, except D11-D16.
Accelerated stability test of lyophilized serotypes 1, 2 and 3 IPV-formulations as shown in Figure 7 (Experiment D) (measured by D-antigen recovery after 1 week incubation at 45 ° C).
D-antigen recovery immediately after lyophilization of serotypes 1, 2 and 3 IPV-formulations as shown in Figure 8 (Experiment E). Stabilizing effects of sorbitol (SOR), sucrose (SUC), mannitol (MAN), monosodium glutamate (MSG), peptone (PEP), and MgCl ₂ are achieved by fast freezing (panel A) and slow freezing (panel B). Was tested.
FIG. 9 Stability of lyophilized serotypes 1, 2 and 3 IPV-formulations as indicated, tested after 1 week incubation at 45 ° C. and tested using fast freezing rates (Panel A) and slow freezing rates (Panel B) ( Experiment E). Only the most promising formulations were shown immediately after freeze drying.
Figure 10 D-antigen recovery immediately after lyophilization of serotypes 1, 2 and 3 IPV-formulations (Experiment F, n = 3). The stabilizing effect of peptone was investigated in IPV-formulations with or without 10% mannitol, containing 10% sorbitol, 10% MSG and 5% MgCl ₂ . All formulations were quickly frozen prior to the drying step of the freeze drying process.
Figure 11 Stability of freeze-dried serotypes 1, 2 and 3 IPV-formulations after 1 week incubation at 45 ° C (Experiment F, n = 3). This experiment investigates whether it is possible to enhance the IPV-formulation with or without 10% mannitol, containing 10% sorbitol, 10% MSG and 5% MgCl ₂ while having accelerated stability with peptone or single amino acid addition .
Recovery of D-antigens immediately after lyophilization of serotype 1, 2 and 3 IPV formulations containing other buffer components as indicated in FIG . 12 (Experiment G). All four formulations listed in Panels A, B, C and D contained 10% sorbitol, 10% MSG, and tested with 10mM McIlvaine, 10mM citrate, 10mM histidine, 10mM HEPES and 10mM phosphate buffer. Control formulations were not dialyzed.
Freeze-dried serotypes 1, 2 and 3 of the IPV-formulations containing other buffer components as shown in FIG. 13 and stability after one week of storage at 45 ° C. (Experiment G). Four formulations containing 10% sorbitol, 10% MSG and MgCl ₂ , with or without mannitol, with or without peptone, 10 mM McIlvaine, 10 mM citrate, 10 mM histidine, 10 mM HEPES and 10 mM phosphate buffer Tested together. Control formulations were not dialyzed and diluted with McIlvaine buffer containing excipients as indicated.
FIG. 14 D-antigen recovery immediately after vacuum drying (V), freeze drying at low freeze rate (L) and freeze drying at high freeze rate (S). The stabilizing effect of the various formulations was compared to the IPV vaccine without negative stabilizer (negative control) and IPV formulated with sucrose or trehalose.
Recovery of D-antigens immediately after lyophilization of serotypes 1, 2 and 3 IPV-formulations as indicated in FIG . 15 .
Figure 16 Stability of serotypes 1, 2 and 3 as indicated after freeze drying and subsequent storage at 45 ° C. for 1 week (measured by D-antigen recovery).

In a first aspect, the present invention relates to a method for preparing a dried formulation of a biopharmaceutical. The method of the present invention provides at least two enhancements: 1) minimizing loss of activity of the biologic upon drying of the formulation (and preferably upon subsequent reconstitution of the dried formulation); And 2) stability, i.e., to increase the shelf life of the dried formulation of the biopharmaceutical, i.e. minimize the loss of activity of the biopharmaceutical upon storage of the dried formulation. The method is preferably a biopharmaceutical; And drying an aqueous solution containing one or more of amino acids, polyols and metal salts.

In the method of the present invention, the solution to be dried comprises a) a biopharmaceutical at a concentration specified below and herein below; b) amino acids at the concentrations specified herein below and specified herein below; c) polyols at the concentrations specified herein below and specified herein below; d) metal salts of concentrations specified hereinbelow and specified herein below; e) water; Optionally, f) a buffer of a pH specified hereinbelow, specified hereinbelow, and as specified hereinbelow; And, optionally, g) comprises, consists essentially of, or consists of additional components at a concentration specified herein below and specified herein below.

Biopharmaceutical

"Biopharmaceutical agent" as used herein refers to a biological agent that is physiologically active, when applied to a mammal, particularly when applied to a human patient, preferably when applied in a pharmaceutically acceptable form. The biological agent is preferably an agent produced by or obtainable from a cell, but a synthetic replica of an agent obtainable from a cell and a chemically modified agent obtainable from a cell are included in the term biological agent. The biological agent may be a protein-based agent, that is, an agent containing proteinaceous substances such as proteins, polypeptides, and peptides. Biological agents may further comprise or consist of nucleic acids, such as, for example, DNA, RNA or nucleic acid analogs.

Preferred biologics are viruses or virions, preferably viruses that infect mammals, preferably viruses that infect humans. The virus may be an envelope virus, but is preferably a non-envelope virus. The term 'virus' as used herein includes wild type viruses (eg, natural isolates) in which they occur naturally, as well as 'man-made' attenuation, mutations and defective viruses. The term 'virus' also refers to a recombinant virus, i.e., a gene therapy vector in which one or more of the gene (s) of interest has been replaced by a defective virus lacking one or more viral genes (parts) and one or more gene (s) of interest. It includes viruses produced using recombinant DNA technology. A preferred virus is an enterovirus (Enterovirus), such as blood or cor virus (Picornavirus), and more preferably poliovirus, nose sakki virus (Coxsackievirus), echo virus (echovirus) and rhinovirus (rhinovirus). Most preferably, the virus is a poliovirus. The poliovirus may be an attenuated poliovirus, but is preferably an inactivated poliovirus (IPV). The poliovirus can also be an inactivated attenuated poliovirus. The biologic may comprise one or more of polio virus serotypes 1, 2 and 3, but preferably the biologic includes all three polio virus serotypes 1, 2 and 3. Suitable strains of serotype 1 poliovirus include, but are not limited to, one or more of the Sabin 1, Mahoney, Brunhilde, CHAT and Cox strains. Suitable strains of serotype 2 poliovirus include, but are not limited to, one or more of Sabin 2, MEF-1 and Lansing strains. Suitable strains of serotype 3 poliovirus include, but are not limited to, one or more of Sabin 3, Saukett H and G, and Leon strains. In a preferred embodiment, the biologic is Salk-IPV containing, for example, an inactivated polio virus Mahoney strain for type 1, an inactivated polio virus MEF-1 strain for type 2 and an inactivated polio virus Saukett strain for type 3 , Or a trivalent inactivated polio vaccine such as sIPV containing the inactivated polio virus Sabin-1, -2 and -3 strains. Methods for inactivating polio virus strains for safe use in vaccines are well known in the art, and include, for example, inactivation with formalin or beta-propiolactone (see, eg, Jonges et al., 2010, J. Clin. Microbiol. 48: 928-940).

In another embodiment, the biologic is a virus or virion of a pneumovirus. The pneumovirus is preferably a Respiratory Syncytial Virus (RSV), more preferably a human or bovine RSV. Human RSV can be a subgroup A or B virus, preferably a clinical isolate, more preferably an isolate that has not been extensively passaged (preferably less than 10, 8, 6 or 5 times) in vitro. Preferably (human or bovine) RSV virus is described in WO 2005/061698 and Widjojoatmodjo et al. (2010, Virol. J., 7: 114), e.g., by mutating within its viral genome, the viral genome does not encode functional G adhesion proteins such as RSV ΔG and RSV ΔG + G virions. Is a virus comprising a viral genome with a deleted or inactivated G attachment protein gene.

The biopharmaceutical is preferably present in an amount in the range of 1 x 10 ⁰ to 1 x 10 ²⁵ raw and / or dead or inactivated particles per ml in the solution to be dried. The number of live particles can be detected, for example, by plaque forming unit cell culture or tissue culture 50% infection dose (CCID ₅₀ or TCID ₅₀ ) and other virological assays suitable for measuring the titer of the formulation. The number of dead or inactivated particles can be detected, for example, using an assay that quantifies the amount of antigen, such as a protein assay, or an assay that detects a hemagglutination unit or a folio D-antigen unit. Preferably the biologic is at least 1 x 10 ¹ , 1 x 10 ² , 1 x 10 ³ , 1 x 10 ³ , 1 x 10 ⁴ , 1 x 10 ⁵ , 1 x 10 ⁶ , 1 x 10 ⁷ per ml in the solution , 1 x 10 ⁸ , 1 x 10 ⁹ or 1 x 10 ¹⁰ raw and / or dead or inactivated particles and / or up to 1 x 10 ²⁴ , 1 x 10 ²³ , 1 x 10 ²² , 1 x 10 ²¹ , 1 x 10 ²⁰ , 1 x 10 ¹⁹ , 1 x 10 ¹⁸ , 1 x 10 ¹⁷ , 1 x 10 ¹⁶ , 1 x 10 ¹⁵ or 1 x 10 ¹⁴ present in amounts of raw and / or dead or inactivated particles.

The amount of biopharmaceutical in the solution to be dried can also be expressed as the weight of the biopharmaceutical per ml of solution. Preferably, the biopharmaceutical is present in a weight / ml ranging from 1 pg / ml to 10 g / ml. More preferably, the biopharmaceutical is in solution in an amount of at least 10 ^-11 , 10 ^-10 , 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 , 10 ^-5 , 10 ^-4 g / ml and / or Up to 10 ^-3 , 10 ^-2 , 10 ^-1 , or 10 ⁰ g / ml. The weight of the biopharmaceutical in solution can be detected, for example, by means known in the art, including protein assays. Thus, the above-mentioned weight of the biopharmaceutical can also be expressed as gram protein per ml to be detected in a suitable protein assay (eg, Bradford assay; Zor and Selinger, 1996, Anal. Biochem. 236: 302-308). .

In a preferred embodiment where the biologic is a poliovirus, the amount of poliovirus in solution is at least 0.01, 0.1, 1.0 or 10 DU / ml, and up to 10,000, 1,000 or 100 DU / ml, where 1 DU is Westdijk et al. [6] It is defined as described by and detected by QC-ELISA. In embodiments where the biopharmaceutical comprises a multivalent poliovirus (vaccine), it is understood that each poliovirus serotype is present in an amount of at least 0.01, 0.1, 1.0 or 10 DU / ml to up to 10,000, 1,000 or 100 DU / ml. .

In a preferred embodiment where the biologic is RSV, the amount of RSV in solution is at least 1 x 10 ¹ , 1 x 10 ² , 1 x 10 ³ , 1 x 10 ³ , 1 x 10 ⁴ TCID ₅₀ / ml, and up to 1 x 10 ²⁵ , 1 x 10 ²⁰ , 1 x 10 ¹⁵ , 1 x 10 ¹² , 1 x 10 ¹⁰ or 1 x 10 ⁹ TCID ₅₀ / ml, TCID ₅₀ for RSV is Widjojoatmodjo et al. (2010, Virol. J., 7: 114).

amino acid

The amino acid in the solution to be dried can be any pharmaceutically acceptable D- or L-amino acid. These amino acids are the 20 standard 'proteinogenic' or 'natural' amino acids (histidine, alanine, isoleucine, arginine, leucine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine , Tryptophan, glycine, valine, proline, tyrosine and serine), as well as non-natural amino acids such as, for example, ornithine, citrulline, selenocysteine, taurine and pyrrolysine. Preferably, the amino acid in the solution to be dried is at least one of glutamate, glutamine, arginine, histidine, asparagine, lysine, leucine and glycine, more preferably at least one of glutamate, arginine and histidine. Accordingly, the solution to be dried may contain a mixture of amino acids. The amino acid (s) in the solution to be dried may be optical D- or L-isomers or mixtures thereof, but preferably the amino acid (s) are L-isomers.

In a preferred embodiment, the amino acid in the solution to be dried is one or more of L-glutamate, L-arginine and L-histidine. In addition, L-glutamate may be in the form of Na-glutamate, peptone, non-animal peptone, vegetable peptone. In addition, L-arginine may be in the form of poly-L-arginine, peptone, non-animal peptone, vegetable peptone. In addition, L-histidine may be in the form of Na-histidine. In addition, L-lysine may be in the form of poly-L-lysine.

In a particularly preferred embodiment, the amino acids in the solution to be dried include glutamate. The glutamate can be one or more of sodium glutamate, potassium glutamate, ammonium glutamate, calcium diglutamate, magnesium diglutamate and glutamic acid. More preferably, glutamate is dissolved in solution in the form of monosodium glutamate (MSG).

The amino acid (s) is preferably present in a concentration in the range of 0.01-20% (w / v) (i.e., weight percent per volume of solution) in the solution to be dried. Thus, preferably, the amino acid (s) is at a concentration of at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 8.0, 9.0 or 9.5% (w / v), or higher in the solution to be dried. Present, and / or the amino acid (s) are present in a solution to be dried at a concentration of 20.0, 17.5, 15.0, 12.5, 11.0 or 10.5% (w / v) or less. Most preferably, the amino acid (s) is present in the solution to be dried at a concentration of about 10% (w / v).

Polyol

The polyol in the solution to be dried is preferably a sugar or sugar alcohol. Preferred sugars include sucrose, trehalose, mannose and dextran. Preferred sugar alcohols include sorbitol, mannitol and maltitol. Preferably, the solution to be dried comprises at least one or more of sorbitol, mannose, mannitol and maltitol, more preferably at least one or more of sorbitol, mannose and mannitol, more preferably at least one or both of sorbitol and mannitol. do. Most preferably, the solution to be dried comprises at least sorbitol as a polyol, and optionally sorbitol together with mannitol.

In one embodiment, the polyol (s) is preferably present in a concentration in the range of 0.1-20% (w / v) (ie, weight percent per volume of solution) in the solution to be dried. Thus, preferably, the polyol (s) is present in a solution to be dried at a concentration of at least 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 8.0, 9.0 or 9.5% (w / v), or higher, and / or The polyol (s) are present in a solution to be dried at a concentration of 20.0, 17.5, 15.0, 12.5, 11.0 or 10.5% (w / v) or less. Most preferably in this embodiment the polyol (s) is present in the solution to be dried at a concentration of about 10% (w / v). Concentrations in this embodiment are suitable, for example, when sorbitol is used as the polyol.

In another embodiment, the polyol (s) is preferably present in the solution to be dried at a concentration in the range of 0.1-40% (w / v) (ie, weight percent per volume of solution). Thus, preferably, the polyol (s) is at a concentration of at least 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 12.5, 15.0, 18.0, 19.0 or 19.5% (w / v), or higher in the solution to be dried. And / or the polyol (s) are present in a solution to be dried at a concentration of 50.0, 40.0, 35.0, 30.0, 25.0, 22.5, 21.0 or 20.5% (w / v) or less. Most preferably in this embodiment the polyol (s) is present in the solution to be dried at a concentration of about 40% (w / v). In this embodiment, the concentration is appropriate when two different polyols are used, for example sorbitol and mannitol. In this case, the weight ratio between the two polyols can range from 1: 100 to 1: 1, including, for example, 1:50, 1:20, 1:10, 1: 5 and 1: 2.

Metal salt

The metal salt dissolved in the solution to be dried may be any pharmaceutically acceptable salt of a divalent or monovalent metal cation. Preferred divalent cations are Ca ⁺⁺ , Mg ⁺⁺ and Zn ⁺⁺ , among which Ca ⁺⁺ and Mg ⁺⁺ are more preferred, and Mg ⁺⁺ is most preferred. Preferred monovalent cations are Li ⁺ , Na ⁺ and K ⁺ , of which Li ⁺ and Na ⁺ are more preferred, and among these, Li ⁺ is most preferred. The counter-anion in the metal salt is preferably not an amino acid, preferably not glutamate or aspartate. More preferably, the counter anion in the metal salt is an inorganic anion. Preferred (No), the anionic counter ion is Cl ^-, SO ₄ ^{2 -} and CO ₃ ^{2 -,} and in the Cl ^- and SO ₄ ^{2 -} and more preferably, in the Cl ^- it is most preferred. The solution may include a mixture of one or more of the metal salts.

The metal salt (s) is preferably present in the solution to be dried to a concentration in the range of 0.005-10% (w / v) (ie, weight percent per volume of solution). Thus, preferably, the metal salt (s) is at a concentration of at least 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, or 4.5% (w / v) or higher in the solution to be dried. And / or the metal salt (s) are present in the solution to be dried to a concentration of 10.0, 8.0, 7.0, 6.0, or 5.5% (w / v) or less. Most preferably, the metal salt (s) are present in the solution to be dried at a concentration of about 5% (w / v).

When the amino acid in the solution to be dried is dissolved in the form of a metal salt of an amino acid such as MSG, the amino acid metal salt is not included in the weight percent of the metal salt (s), but is included in the weight percent of the amino acid (s), thereby Accordingly, it is understood that the weight of the metal counter cation is included in the weight of the amino acid.

buffer

The solution to be dried preferably has a neutral pH, such as a pH in the range of 6.0-8.0 or 6.5-7.5 or a pH of about 7.0. The solution to be dried preferably contains a buffer to maintain the pH at the values indicated above. In principle, any pharmaceutically acceptable buffer, preferably a buffer having an effective buffering capacity within the range of the pH values indicated above, can be used in the solution to be dried. The buffer is preferably present at a concentration in the range of 0.5-100 mM, more preferably in the range 1-50 mM, and most preferably in the range of 2-20 mM, such as about 10 mM.

Buffers suitable for use in the solution to be dried include McIlvaine buffer (reference example), citrate buffer, phosphate buffer, HEPES buffer and histidine buffer.

water

The water used to prepare the solution to be dried is preferably ultrapure water, preferably pyrogen-free water such as water for injection.

The concentration of the components (eg, excipients) in the solution to be dried of the present invention is generally expressed herein as weight percent (w / v) per volume of the solution. It is understood that the relationship of solute (eg excipient) to solvent is expressed in grams of solute per liter of total solution. For example, 50 g of glucose in 1 L of solution is considered a 5% w / v solution.

In a preferred embodiment, the solution to be dried is a biopharmaceutical, 5-20% (w / v) sorbitol, 5-20% (w / v) monosodium glutamate, 2-10% (w) in an amount as defined herein above. / v) of a magnesium salt, preferably MgCl ₂ , and / or MgSO ₄ , optionally 5-20% (w / v) mannitol, and optionally buffering the solution at neutral pH as shown herein 2- It consists essentially of 50 mM pharmaceutically acceptable buffer.

dry

In the method of the present invention, the aqueous solution containing the biologic is dried. Any known drying method can be used. The drying method can be, for example, spray drying, air drying, coating, foam-drying, desiccation, vacuum drying, vacuum / freeze drying or freeze drying, all of which are known to those skilled in the art. have. In a preferred embodiment, the drying method is vacuum drying or freeze drying, of which freeze drying or freeze drying is more preferred.

In one embodiment of the freeze drying process, the solution to be dried is preferably first frozen to an initial freeze drying or storage temperature of -50 ° C, -40 ° C, -30 ° C, -20 ° C or -10 ° C or less. The preferred initial storage temperature is -50 ° C or -40 ° C or less. The solution to be dried can be primary frozen by immediately placing the solution (container / vial containing) into storage with an initial storage temperature as described above. Alternatively, the solution to be dried is preferably placed by placing the solution (container / vial containing) in a preservation having a temperature of 0 ° C. or higher, such as 2, 4 or 6 ° C., and then lowering the temperature, Can be slowly frozen by slowly freezing the solution to the initial freeze drying temperature as described above by lowering the temperature at a rate of about 0.5, 1 or 2 ° C. per minute. The solution to be dried may be provided at a pressure of 100 microbars or less. When the set pressure is reached, the storage temperature can be increased to a higher temperature. The storage temperature can be increased to a temperature of 5, 10 or 15 ° C. above the initial freeze drying temperature, for example at a rate of 0.5, 1 or 2 ° C. per minute. The primary drying step is preferably terminated when no increase in pressure is measured in the chamber. Preferably, at this moment the storage temperature can be increased to, for example, 5, 10, 15, 20 or 25 ° C at a rate of 0.01, 0.02 or 0.05 ° C per minute, optionally in one or more steps. Can be increased together. During the second drying step, the temperature is preferably maintained at that value until no pressure increase is detected. Preferred freeze drying processes are described in the Examples herein.

In one implementation of the vacuum-drying process, the solution to be dried is preferably present at a temperature in the range of about 5-25 ° C, such as room temperature, or more preferably, in a range of about 10-20 ° C, such as about 15 ° C. The pressure is then reduced to pressures less than 1, 0.5, 0.2, 0.1 and 0.05 mbar. To suppress freezing of the solution, the temperature of the solution to be dried under reduced pressure can be reduced to a temperature below 0 ° C. but above the eutectic temperature of the solution. If the pressure rise in the chamber is not measured, the temperature of the solution can be increased, for example, at a rate of 0.01, 0.02 or 0.05 ° C per minute, for example, 5, 10, 15, 20 or 25 ° C, Optionally, this can be increased in one or more steps. The temperature is preferably maintained at that value until no pressure increase is detected. Preferred vacuum drying processes are described in the Examples herein.

In a second aspect, the invention relates to a dried or dried formulation of a biopharmaceutical obtainable or obtained by the method according to the invention as described above.

The method of the present invention for preparing a dried formulation of a biopharmaceutical is preferably aimed at minimizing loss of activity of the biopharmaceutical upon drying the formulation. Preferably, the method of the invention as well as the formulation itself is also aimed at minimizing the loss of activity of the biopharmaceutical upon subsequent storage of the dried formulation obtainable with the method of the invention. Accordingly, the method of the invention preferably occurs in a stable formulation of a biopharmaceutical, i.e., preferably under refrigerated conditions (e.g. 2-10 ° C), at room temperature (e.g. 18-25 ° C), or in tropics. It is a method of preparing formulations with prolonged or extended shelf life, even at elevated temperatures (eg 32-45 ° C.).

It is understood that loss or inactivation of the activity of a biopharmaceutical includes both chemical pathways (oxidation, hydrolysis or enzymatic action), as well as loss of activity due to physical pathways (denaturation, aggregation, gelation). Preferably, the loss of activity of the biopharmaceutical does not exceed an acceptable level. That is, after drying and / or subsequent storage, at least the level of biological activity or viability sufficient for the intended commercial therapeutic and / or diagnostic application of the biologic is maintained and / or the level of the original function or structure is maintained.

Depending on the nature of the biopharmaceutical, the skilled artisan will know which assay is used to evaluate the activity of the biopharmaceutical. The activity of a biopharmaceutical can be expressed in its viable activity, for example as in the case of an active live virus, or the activity can be expressed in enzymatic or biological activity, which can be measured by appropriate assays known to those skilled in the art. Can be. In another example, the activity of a biopharmaceutical can be related to the physical and chemical integrity of the biopharmaceutical, and can be detected by evaluating the structure and / or function of the biopharmaceutical. Antigen structure is preferably evaluated by ELISA or Biacor analysis. Secondary and tertiary structures are preferably evaluated by UV-, fluorescence, Fourier Transformed Infra Red (FTIR) and / or circular dichroism spectroscopy. Immunogenicity is preferably assessed by in vivo test analysis using animal models (eg, rat, rat, cotton rat, ferret or rabbit). Rabbits are the preferred model for, for example, polio virus, and cotton rats are the preferred model for RSV.

In a preferred embodiment of the method of the invention, drying of the solution (to be dried) is followed by at least 50, 60, 70, 75 of the biological activity of the dried formulation in the solution prior to drying upon rehydration (preferably immediately) after drying. Be sure to hold 80, 85, 90 or 95%. If more than one biologic is present in the formulation, the difference in loss of activity for other biologics is less than 50, 60, 70, 75, 80, 85, 90 or 95%, and thus the organism with the highest loss of activity The retention activity of the drug is expressed as a percentage of the retention activity of the biologic with the lowest loss set to 100%.

In another preferred embodiment of the invention, the dried formulation is stored at 45 ° C. for at least one week and upon rehydration of the dried formulation, at least 50, 60, 70, 75 of the biological activity present in the solution in the dry gun. 80, 85, 90 or 95%. When one or more biologics are present in the formulation, the difference in loss of activity for other biologics is preferably less than 50, 60, 70, 75, 80, 85, 90 or 95%, thus leading to the highest loss of activity. The retention activity of the biologic possessed is expressed as a percentage of the retention activity of the biologic having the lowest loss set to 100%. Preferably also stored at 37 ° C. or 45 ° C. after storage as described above after 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more Shows the percentage of activity.

In a third aspect, the present invention relates to the formulation of a biopharmaceutical obtainable or obtained by the method according to the present invention as described herein above as being used as a medicament. For use as a medicament, the formulation can be used as a dried formulation or can be reconstituted, for example, by dissolving the dried formulation using water as described above. The formulation is preferably reconstituted in its original volume, ie before drying. Preferably, the formulation is a formulation for inducing an immune response (in an individual) against an infectious disease or tumor. The subject or subject to which the formulation of the present invention is administered may be a human, but may also be an animal, such as a breeding animal or pet, including, for example, mammals, birds, livestock, poultry, cattles, and cattle. More preferably, the formulation is for vaccination against an infectious disease or tumor. Accordingly, the formulation is preferably a formulation for the prevention or treatment of infectious diseases or tumors. In another embodiment, the present invention provides for the preparation of a medicament that induces an immune response in a form obtainable or obtained according to the method of the present invention as described herein above, for immunization against an infectious disease or tumor, and / or It relates to use for the prevention or treatment of infectious diseases or tumors. In another embodiment, the present invention relates to a method of inducing an immune response to an infectious agent or tumor by administering an effective amount of a formulation to a subject in need of induction of an immune response to the infectious agent or tumor. The immune response is preferably directed against the antigen in the biologic. The antigen is preferably an antigen of a pathogen causing an infectious disease or an antigen of a tumor, or an antigen that induces an immune response against the pathogen or the tumor. The pathogen is preferably a virus as defined herein above. The formulations of the invention can be administered with or without reconstitution via intranasal, parenteral, intramuscular, subcutaneous and / or transdermal routes.

In the present specification and claims, the verb “include” and its verb conjugation form are used as a non-limiting standpoint, meaning that items following the letter are included, but items not specifically mentioned are not excluded. In addition, reference to a component that is modified with the indefinite article “one (a or an)” means that one or more components are required, unless the circumstances clearly require one or only one component to be present. It does not rule out the possibilities that exist. The indefinite article “one (a or an)” thus generally means “at least one”.

All patents and literature references cited in this specification are hereby incorporated by reference in their entirety.

The following examples are provided only to illustrate the present invention, and are not intended to limit the scope of the present invention in any way.

Example

1. Materials and Methods

1.1 Materials

A trivalent inactivated polio vaccine (Salk-IPV) containing an inactivated, Mahoney strain for Type 1, MEF for Type 2 and Saukett for Type 3 was obtained from RIVM-Vaccinology's Process Development Department (Bilthoven, Netherlands) Did. Salk-IPV trivalent bulk (10x) was formulated in a 10-fold concentrated 40-8-32 DU / single human dose (1 ml). The concentration of IPV 05-126B bulk used in this test was measured at 411-90-314 DU using the QC-ELISA described by Westdijk et al. [6].

Excipients Sucrose, D-sorbitol, D-trehalose dihydrate, D-glucose monohydrate, mannitol, L-glutamic monosodium salt monohydrate (herein glutamate, sodium glutamate, monosodium glutamate or MSGra Sigma (St. Louis), myo-inositol, D-rapinose, hydroxy ethyl starch, glycine, L-proline, L-leucine, calcium chloride dihydrate, maltitol, magnesium chloride hexahydrate, lithium chloride and ovalbumin. , MO). Peptone (vegetable), dextran (6 kDa, Leuconostoc ssp), L-histidine, L-alanine, zinc chloride, calcium lactobionate monohydrate were purchased from Fluka (Buchs, Switzerland). Lactitol® (Lacty®-M) was purchased from Purac Biochem (Gorinchem, Netherlands), L-arginine (EP, non-animal origin) and Tween80 from Merck (Darmstadt, Germany), polyvinylpyrrolidone 25 (PVP, 29 kDa) ) Was purchased from Serva Feinbiochemica GmbH (Heidelberg, Germany), Sol-U-Pro, hydrolyzed focin gelatin was purchased from Dynagel Inc. (Calumet City, IL), and Ficoll was purchased from Pharmacia (Uppsala, Sweden). Purchased from. As buffer components, sodium dihydrogen phosphate dihydrate (NaH ₂ PO4), sodium chloride (NaCl), potassium dihydrogen phosphate (KH ₂ PO ₄ ) and EDTA purchased from Merck were used. Trisodium citrate dihydrate, citric acid and HEPES were purchased from Sigma-Aldrich (St. Louis, MO), and disodium hydrogen phosphate dihydrate (Na ₂ HPO ₄ ) was purchased from Fluka (Buchs, Switzerland). All excipients were used with reagent quality or higher grade.

To prepare a 10 mM McIlvaine buffer, 10 mM citric acid was added to 10 mM Na ₂ HPO ₄ at a ratio of 1: 6 and the pH value was adjusted to 7.0. For the 10 mM citrate buffer, the components trisodium citrate dihydrate (10 mM) and citric acid (10 mM) were mixed together until pH 7.0. The 10 mM phosphate buffer at pH 7.0 consisted of 10 mM KH ₂ PO ₄ and 10 mM Na ₂ HPO ₄ . 10 mM HEPES and 10 mM histidine buffer were prepared by weighing and dissolving the buffer components and then adjusting the pH value to 7.0 using HCl and / or NaOH.

1.2 Method

1.2.1 Dialysis

Unless otherwise indicated, trivalent IPV bulk material was used with a 10kMa molecular weight cut-off, low-binding regenerated cellulose membrane dialysis cassette (Slide-A-Lyzer®, Pierce, Thermo Scientific, Rockford, IL) using a 10mM McIlvaine buffer ( dialysis against pH 7.0) to replace the buffer component of IPV bulk (M199 medium).

1.2.2 Solution to be dried

All excipients were dissolved in McIlvaine buffer at a concentration twice the final concentration indicated. The dialyzed IPV was mixed equally with the formulation to be tested. Subsequently a 2 ml glass injection vial (Muller + Muller, Holzminden, Germany) was filled with 0.2 ml of IPV-excipient mixture and a 13 mm pre-dried (overnight at 90 ° C.) rubber stopper (type V9250, Helvoet Pharma, Alken, Belgium).

1.2.3 Freeze drying and vacuum drying process

For freeze drying, the filled and half-closed vials are frozen at -50 ° C by slowing the temperature at a rate of 1 ° C / min at a storage temperature of -50 ° C (quick freezing) or a storage temperature of 4 ° C (slow freezing) ) Leybold GT4 freeze dryer or Zirbus pilot / laboratory freeze dryer sublimator 2-3-3. The vial was maintained at a temperature of -50 ° C for 2 hours. For the first drying step, the storage temperature was increased to -45 ° C at 0.1 ° C / min, then increased to -40 ° C at 0.02 ° C / min, and then incubated for 42 hours. The second drying step was increased to 10 ° C at 0.02 ° C / min, and then cultured at 10 ° C for 8 hours. Thereafter, the storage temperature was increased to 25 ° C at 0.02 ° C / min.

For vacuum drying, the filled and half-closed vials were loaded into a Zirbus freeze dryer sublimator 2-3-3 at a storage temperature of 15 ° C. and held at that temperature for 10 minutes. The chamber pressure was reduced to 1 mbar in a ramping step of 15 minutes at various speeds (1 mbar / min, 0.3 mbar / min, 0.1 mbar / min), starting at 25 mbar chamber pressure. The temperature was reduced to −10 ° C. for 1 hour at 0.05 mbar and 1 hour at 0.03 mbar, without causing freezing of the formulation (product temperature above the eutectic temperature of the formulation). Subsequently, the storage temperature was increased to 0.05 ° C / min to 30 ° C. At the end of the cycle, the vial was closed under vacuum, sealed with an aluminum lid and stored at 4 ° C. until analysis. Table 1 shows examples of storage temperature and chamber pressure during the vacuum drying process.

T _shelf
(℃) term
(min) pressure
(mbar) FT01 15 10 - D01 15 15 25 D02 15 15 10 D03 15 15 5 D04 15 15 3 D05 15 15 One D06 -10 60 0.05 D07 -10 60 0.03 D08 -5 120 0.03 D09 5 120 0.03 D10 10 120 0.03 P01 20 240 - P02 30 240 - P03 4 60 -

1.2.4 D-antigen ELISA

Polystyrene 96-well microtiter plates were coated overnight at room temperature with serotype-specific bovine anti-policy serum (RIVM, Bilthoven, The Netherlands). After washing with 0.1% Tween20 (washing buffer) in PBS, IPV-formulation of a single dilution diluted in IPV reference standard and assay buffer (PBS containing 0.5% Protifar and 0.1% Tween20) in 2 dilutions was added. (100 μl / well in two). Plates were incubated at 37 ° C for 30 minutes under gentle agitation, washed extensively, serotype-specific monoclonal mouse antibody (mab 3-4-E4 (type 1), 3-14-4 (type 2), 1 A mixture of -12-9 (type 3), all RIVM (Bilthoven, Netherlands) and HRP-labeled anti-mouse IgG (GE Healthcare, Buckinghamshire, UK) was added. Subsequently, the plates were incubated at 37 ° C for 30 minutes under gentle stirring. Plates were washed extensively, ELISA HighLight signal reagent (Zomerbloemen BV, Zeist, Netherlands) was added, and chemiluminescence was measured for 10-15 minutes using a photometer (Berthold Centro LB960).

1.2.5 Moisture-content analysis

The moisture content was specified using a Karl Fischer coulometric titrator (Model CA-06 Moisture meter, Mitsubishi). The principle of moisture retention measurement by Karl Fischer method is based on the fact that iodine and sulfite react only in the presence of water. Samples were weighed and then reconstituted in Karl Fischer reagent, Hydranal Coulomat A (Fluka, Buchs, Switzerland). The reconstituted sample was collected by syringe and injected into a titration container. Each vial was measured three times. The empty vial is weighed and the water content is based on the water content measured by a titration device, the weight of the freeze-dried product in the vial, the reconstitution capacity of the reagent, the titration capacity and the water content of the blank titration. Was measured.

1.2.6 Differential Scanning Calorimetry ( DSC )

The thermodynamic behavior of the formulation was measured by differential scanning calorimetry (DSC), a method of measuring the temperature and heat flow associated with phase transfer in a material as a function of time and temperature. The lyophilized formulation was charged to an aluminum DSC pan and applied to a controlled temperature program in a differential scanning calorimetry (DSC Q100, TA Instruments). The sample was heated from 0 ° C to 150 ° C at a rate of 20 ° C / min and purged with nitrogen gas (50ml / min). The glass transition temperature (Tg) was measured as a discontinuous midpoint in the heat flow curve using software (Universal Analysis 2000, TA Instruments).

2. Results

2.1 Various during freeze drying IPV  Stabilization of subtype

In the first experiment (Experiment A), four well-known stabilized saccharides / polyols (sucrose, trehalose, mannitol and dextran), as well as sodium chloride were evaluated for their stabilization potential (Figure 1). The trial was set up with the design of an experimental approach to obtain the optimal formulation for freeze drying of IPV.

Various IPV-formulations were lyophilized as described above (section 1.2.3). The freeze-dried cake was reconstituted by adding the same amount of water as the starting capacity, and the D-antigen recovery was measured by ELISA (section 1.2.4). The recovery rate is expressed as a percentage of the D-antigen content in the liquid formulation measured prior to freeze drying.

Trivalent IPV formulations containing no additives and diluted 1: 1 with IPV and McIlvaine buffers showed <10% recovery for all serotypes after lyophilization (Figure 1; A1). Type 2 IPV was found to be the most stable serotype in all formulations with a maximum D-antigen recovery of ± 80%. Dextran has been shown to negatively affect the D-antigen recovery of lyophilized IPV formulations, especially Types 1 and 3. For serotypes 1, 2 and 3, formulations containing sucrose and / or trehalose with best results with mannitol with maximum recovery of ± 55%, ± 85% and ± 50%, respectively (Figure 1; Formulations A3, A4, A12 and A16). The addition of NaCl did not have a positive effect on the recovery of IPV after freeze drying.

This first pilot experiment clearly demonstrates the complexity of lyophilization of a trivalent polio vaccine where each IPV serotype favors each stabilizer. Formulations containing 10% mannitol Type 1 and Type 3 preferred the presence of high concentration sucrose free of trehalose, whereas Type 2 preferred high concentrations of trehalose free of sucrose (Figure 2). .

In the next experiment (Experiment B), the stabilization potential of a mixture of glutamate, saccharide and polymer was investigated. Various combinations of excipients sucrose, trehalose, monosodium glutamate (MSG), hydroxyethyl starch (HES) and NaCl were investigated. Trivalent IPV using MSG based formulations with disaccharides, sucrose and / or trehalose freeze-dried D- of 50-60%, 70-95% and 50-65% for the three serotypes, respectively. Antigen recovery was shown (Fig. 3; Formulations B4, B7, B9, B12, B13). Formulations based on only 8% HES or 63 mM NaCl did not protect IPV during freeze drying (FIG. 3; B5 and B10). Addition of NaCl to the formulation containing sucrose showed a 5-10% increase in D-antigen recovery after lyophilization (Figure 3; B2 and B16).

2.2 Effect of drying process and formulation

Next, the test results of the typical formulation used for drying the biopharmaceutical with respect to the drying process are shown. Formulations containing trivalent IPV are vacuum dried (freeze drying method), freeze drying using a fast freezing step (by placing the product directly at a pre-cooled storage temperature of 50 ° C) and freeze drying using a slow freezing step (4 The product was placed at a storage temperature of ° C, and dried by freezing at -50 ° C). As shown in Figure 4, for example, standard formulations based on sucrose or trehalose partially protect IPV when vacuum dried. However, these formulations do not provide protection when lyophilized.

2.3 Screening of excipients

In the next experiment (Experiment C), various formulations containing sorbitol, mannitol, sucrose and / or MSG along with some amino acids, proteins / peptides or other stabilizers were tested (Table 2). To investigate the effect of salts in the lyophilized IPV-formulation, C-formulations were also tested by adding 125 mM NaCl. The effect of NaCl on D-antigen recovery was not clearly observed (data omitted). Initial observations of antigenic results immediately after lyophilization showed that formulation C13 containing 5% sorbitol, 5% peptone and 1% lithium chloride (LiCl) was ± 85 for all serotypes, types 1, 2 and 3, respectively. It was clear that the highest recovery rates of%, ± 100% and ± 85% were shown (Fig. 5). The replacement of the LiCl-compound in 1.8% MgCl ₂ resulted in ± 5%, ± 20% and 15% reduction in D-antigen recovery for the three serotypes. However, these formulations showed a relatively high residual moisture content, 2.1% for MgCl ₂ -containing formulations and 6.2% for formulations containing LiCl. The addition of a very small amount of surfactant Tween 80 to a formulation containing sucrose, trehalose and amino acids glycine and lysine increased the D-antigen recovery by 5-15% (Figure 5; C8, C9). Formulations based on sorbitol, mannitol and MSG (C2, C3, C4 and C7) showed recovery rates of at least 65%, 80% and 70% for Types 1, 2 and 3, respectively.

In this test, the combination of sorbitol, peptone and salt LiCl or MgCl ₂ appeared to have a positive effect on D-antigen recovery immediately after freeze drying of IPV. Another notable formulation is a mixture of sorbitol, mannitol and MSG, which has shown that the presence of polyols with MSG stabilizes IPV during freeze drying.

The dielectric transition temperature of the lyophilized formulations was measured (Table 2), but did not show a clear correlation with the D-antigen recovery rate. Formulations containing ovalbumin and peptone showed the best glass transition temperature.

Lyophilized IPV-formulation composition (Experiment C). Residual moisture content (RMC) was measured by Karl Fischer and Tg of the dried cake was measured by DSC.
SOR
MAN
SUC
MSG
sugars/
Polyol
amino acid
protein
Other
RMC  (%)
T _g
(℃) C1 - - - - - - - - 3.6 n.d. C2 7% 7% - 2% - 2% glycine 7% ovalbumin - 0.3 37.2 C3 7% 7% - 2% - 2% glycine 7% ovalbumin 1 mM EDTA n.d. 38.3 C4
7% 7% - 2% - 2% glycine - - 1.9 53.5 C5

- - 3% - 3% dextran
3% myo-inositol - 3% ovalbumin - 1.1 54.9 C6

5% 5% 5% - - 2% glycine
3% Lysine
3% L-Arg - - 1.0 37.1 C7
5% 5% 5% 3% - 2% glycine
3% lysine - - 3.3 37.2 C8
- - 5% - 5% Trehalose 3% lysine
3% Alanine - - 0.5 32.3 C9
- - 5% - 5% Trehalose 3% lysine
3% Alanine - 0.01% Tween80 0.5 34.1 C10
- - 5% - - 3% lysine
3% Alanine - 3% Ca-lactobionate 1.0 31.2 C11
- - 5% - - 3% lysine
3% alanine 3% Rec. gelatin - 0.3 35.7 C12 5% - - - - - 5% peptone 1.8% MgCl ₂ 2.1 44.7 C13 5% - - - - - 5% peptone 1% LiCl 6.2 n.d. C14 - - 5% - 5% Trehalose - 5% peptone - 0.5 35.6

Based on these findings, a new screening experiment (Experiment D) was designed. Since the most promising recovery rates were observed with formulations based on sorbitol, peptone and Mg or Li-chloride, experiments based on 10% sorbitol, 5% peptone and 125 mM NaCl were designed. Ovalbumin was of animal origin and was excluded as an undesirable excipient in vaccines for human use. To gain more insight from the IPV stabilization mechanism of several excipients, formulations containing 10% sorbitol, 5% peptone and 125mM NaCl are sugars / polyols, amino acids (instead of 5% peptone), salts or other agents such as surfactants or proteins. It was mixed with a stabilizer. The formulation containing sorbitol, NaCl and 1% histidine showed a recovery rate of 90-100% immediately after the freeze-drying process (Fig. 6; D11). As initially observed, MgCl ₂ -containing formulations exhibited auspicious stabilizing ability during freeze drying, which was similar to calcium chloride (CaCl ₂ )-and lithium chloride (LiCl) -containing IPV formulations (FIG. 6; D17-). 19). During the screening step, accelerated stability was evaluated to screen for excipients with their ability to provide stable lyophilized products as well as to provide stability after subsequent storage at high temperatures.

2.4 Accelerated Stability Test

Although some formulations showed acceptable D-antigen recovery after incubation at 45 ° C. for 1 week immediately after freeze drying, the D-antigen recovery of these 4 formulations was 30 for serotypes 1, 2 and 3, respectively. %, 60% and 10% or less (Fig. 7). Formulations containing 5% myo-inositol showed the highest recovery after incubation at 45 ° C., but still a decrease of ± 40%, 10% and 30% was observed for the three serotypes, where also IPV type 2 was It was found to be the most stable serotype (FIG. 7; D5). When stored at 45 ° C. for 1 week, formulations containing 1% lactitol showed a D-antigen recovery of less than 10% for serotype 2, and unfortunately serotypes 1 and 3 showed a decrease of> 30%. (FIG. 7; D7).

To further investigate the combination of sorbitol, mannitol, MSG and the stabilization potential of peptone with MgCl ₂ , an experimental setup of a new design was performed to detect the relationship between various excipients and D-antigen recovery after freeze drying. The following variables were included in Experiment E: 0/10% sorbitol, 0/10% sucrose, 0/10% mannitol, 0/10% MSG, 0/5% peptone and / or 0/5% MgCl _2. and The freezing rate was measured in this experiment. Slow freezing means that the vial is placed on a shelf at 4 ° C. and subsequently cooled to −50 ° C. at a rate of 0.1 ° C./min. Fast freezing means the vial is pre-cooled at −50 ° C. It means being placed directly on. The results are shown in Figure 8a for fast freezing rates and in Figure 8b for slow freezing rates.

As a result of first observing the D-antigen recovery after lyophilization, a fast freeze formulation containing MSG and MgCl ₂ with sugars / polyols showed the highest recovery of ± 80-90% for all serotypes (FIG. 8A; E22-24, E29-32). With regard to slow freezing samples, the MgCl ₂ -containing formulations showed 75-90% recovery for Types 1 and 3 (Figure 8B; E54-57, E63-66). However, for serotype 2, only the formulation containing peptone and saccharide (s) showed a recovery rate of ± 70% (Fig. 8b, E43-48). To demonstrate the reproducibility of the experiments, formulations H33 and H66 were tested three times and showed <10% standard deviation for all serotypes. From a comparison of the D-antigen recovery rates of these two formulations immediately after lyophilization (FIG. 8) or after one week incubation at 45 ° C. (FIG. 9), it is clear that the freezing rate does not significantly affect the recovery rate of these IPV formulations.

Experiment E was established based on the 'Design of Experiment' using "Modde" software (Umetrics). The recovery rate of the D-antigen after freeze drying, as well as the recovery rate after freeze drying and subsequent storage at 37 ° C or 45 ° C, was used as output in the experimental design. The output was adjusted as a function of the formulation and placed in the model using Modde. This showed which formulation parameters affected lyophilization and recovery after storage (data omitted).

The most important formulation parameters for each virus serotype are listed in Tables 3-5.

Table 3 Type 1 D-antigen recovery Formulation parameters After freeze drying
% After storage at 37 ℃
% After storage at 45 ℃
% MSG 9 10 7 Sorbitol 8 6 4 MgCl ₂ 7 6 3 peptone 6 4 Mannitol 4 6 MSG * MgCl ₂ 4

Table 4 Recovery rate of type 2 D-antigen Formulation parameters After freeze drying
% After storage at 37 ℃
% After storage at 45 ℃
% MSG 7 9 9 Sorbitol 7 7 5 MgCl ₂ peptone 10 10 Mannitol 3 4 4 Sucrose 2

Table 5 Recovery rate of type 3 D-antigen Formulation parameters After freeze drying
% After storage at 37 ℃
% After storage at 45 ℃
% MSG 14 10 10 Sorbitol 8 7 7 MgCl ₂ 4 4 4 peptone 4 12 12 Mannitol 6 5 5 Sucrose 4 One One MSG * MgCl ₂ 5 5

2.5 Substitution of peptone

Since peptone was shown to stabilize lyophilized IPV during subsequent storage at 45 ° C, it plays a role of peptone in the formulation containing 10% sorbitol, 5% MSG and 5% MgCl ₂ and the same formulation mixed with 10% mannitol. An experiment was conducted to investigate. No significant difference was found when 10% mannitol was added to the formulation containing sorbitol, MSG and MgCl ₂ . Addition of 5% peptone did not affect the antigenicity of both formulations (Figure 10).

To find out if the addition of a single amino acid could replace the stabilizing role of peptone during subsequent storage of lyophilized IPV, amino acids were added to formulations containing sorbitol, MSG and MgCl ₂ and with or without mannitol. After one week incubation at 45 ° C., neither peptone nor added amino acids showed improved stability of D-antigen recovery compared to control formulations containing 10% sorbitol, 10% MSG and 5% MgCl ₂ (FIG. 11, F1.0-F1.5). In the presence of mannitol, the stabilizing ability of peptone appeared only for Type 3, but the D-antigen recovery was improved by +/- 15% compared to the control sample. However, a significantly reduced recovery rate (p <0.001) was found for Type 1, although not significant, Type 2 also exhibited a ± 10% lower recovery rate (FIG. 11; F2.0, F2.1). For all formulations containing sorbitol, MSG and MgCl ₂ , a ± 10% reduction in D-antigen content was seen for Type 2, where the addition of mannitol was found to be stable for Type 2 during accelerated stability. Arginine appeared to have stabilizing potential in formulations containing sorbitol, MSG, MgCl ₂ and mannitol (FIG. 11, F2.3), and D-antigen recovery for Types 1 and 3 after culture at 45 ° C. was ± 15, respectively. % And ± 30%. This was comparable to stabilization with an almost identical formulation containing peptone instead of arginine (FIG. 11, F2.1).

Because unspecified excipients such as peptone are undesirable for human vaccines, possible alternatives to peptone that can stabilize IPV during storage have been investigated. Mass spectrometry and analysis by HPLC showed the most abundant amino acids present in peptone (data omitted). Addition of several single amino acids to the formulation containing sorbitol, MSG and MgCl ₂ did not improve stability at 45 ° C. compared to the control formulation. Peptones have been shown to stabilize serotype 3 in formulations containing sorbitol, MSG, MgCl ₂ and mannitol, and arginine can improve the stability of both serotypes 1 and 3. Serotype 2 already showed complete maintenance of D-antigen recovery in the control formulation during accelerated stability. The exact composition of peptone is difficult to detect, but the amino acid quantification by reverse-phase HPLC using non-hydrolyzed peptone and chemically hydrolyzed peptone is that peptone is composed of both single amino acid and peptide, but more than 90% w / w of peptone is defined Remains unsuccessful. Since peptones appear to increase the glass transition temperature of the investigated IPV-formulation, it may be possible to replace peptone with excipients with high Tg such as sucrose or trehalose.

The glass transition temperatures (Tg 'and Tg) of IPV-formulations with or without mannitol, containing sorbitol, MSG and MgCl ₂ , were measured by DSC. The effect of peptone on the glass transition of these formulations was investigated. Single measurements are shown. 10% Sorbitol + 10% MSG  + 5% MgCl ₂ 10% Sorbitol + 10% MSG  + 5% MgCl ₂
+ 10% Mannitol T _g ' ℃) T _g (℃) T _g ' (℃) T _g (℃) Control -47.9 35.2 -44.4 38.8 + 5% peptone -44.1 39.8 -42.8 48.7

Previous experiments did not produce a valuable substitute for peptone, and formulations containing sorbitol, MSG, and MgCl ₂ , with or without mannitol, were found to provide best results even after accelerated stability testing. During this test, all formulations were prepared with McIlvaine buffer, which is known as a suitable buffer for freeze drying of IPV [52]. To further optimize the formulation, buffer screening was performed using a buffer frequently used for lyophilization of biologics. The IPV batch in each buffer was prepared by dialysis, and the undialysis IPV was used as a control.

Formulations containing sorbitol, MSG and MgCl ₂ showed recovery rates of ± 95%, ± 85% and 90% for the three serotypes immediately after lyophilization (FIG. 12A; G1.0-G1.5). After the addition of peptone, the recovery rates of D-antigens of Types 1 and 3 were reduced by 10-15%, and Type 2 exhibited similar recovery rates compared to formulations containing no peptone (FIG. 12B). McIlvaine buffer showed a 10-15% lower recovery rate for serotype 2 compared to other buffer components. Histidine buffer has been shown to increase the D-antigen yield of IPV after lyophilization of IPV formulations containing sorbitol, MSG and MgCl ₂ (recovery> 95% for Types 1 and 3; FIGS. 12A, G1.3) . After lyophilization, a D-antigen recovery of 88% was achieved for type 2 when phosphate buffer was used, and a 73% recovery for type 2 was obtained in McIlvaine buffer (FIG. 12A, G1.5).

Addition of mannitol to formulations containing sorbitol, MSG, MgCl ₂ and no peptone, serotype 2 favored the presence of mannitol in the formulation during freeze drying, as a recovery of 85-100% was observed for type 2 It was shown (Fig. 12C). For IPV-formulations containing sorbitol, MSG, MgCl ₂ and mannitol, a recovery rate of 80-90% for type 1 and ± 80% for type 3 was observed. The addition of peptone showed a ± 10% reduction in the recovery rates of Types 1 and 3, and a similar D-antigen content for Type 2 after freeze drying (FIG. 12D).

2.6 Effect of Buffer Components

Accelerated stability experiments showed formulation-dependent differences between the buffers used. The control formulation showed a high recovery rate immediately after lyophilization, but after one week orientation at 45 ° C., the peptone-deficient control formulation had ± 40%, ± 50% and <15% of serotypes 1, 2 and 3, respectively. Recovery rates are shown (Fig. 13; G1.0 and G3.0). In the control formulation, the peptone had a D-antigen recovery of ± 25% and ± 15%, instead of> 90% and 80%, respectively, for formulations containing or without mannitol after storage at 45 ° C. In the case of a decrease in, it showed a clear stability improvement of types 1 and 2 (Fig. 13; G2.0 and G4.0). For formulations containing sorbitol, MSG and MgCl ₂ , the highest recovery was observed in HEPES buffer, D-antigen content of ± 80%, ± 85% and ± 60% for the 3 serotypes after freeze drying and storage. . This means that during accelerated stability testing for Types 1, 2 and 3, the D-antigen recovery rates showed only 10%, 0% and 30% reduction, respectively. Better results using citrate buffer for Type 3 only, a decrease of ± 20% was observed, maintaining a recovery rate of 79% (FIG. 13A; G1.2). The addition of peptone showed a total recovery rate of less than 15-25% in all serotypes compared to the same formulation and buffer except that peptone was not added (FIG. 13A). When peptone was added to the formulation containing sorbitol, MSG, MgCl ₂ and mannitol, it clearly showed improved stability. Other buffer formulations without peptone showed maximum recovery rates of ± 55%, ± 75% and ± 45%, and the same buffers with peptone addition showed recovery rates of ± 50%, ± 80% and ± 55%. (FIG. 13B). Formulations based on phosphate buffers containing the excipients sorbitol, MSG, MgCl ₂ , mannitol and peptone are the most stable formulations with reduced recovery of ± 30% for type 1 and only ± 10% for type 2 after incubation at 45 ° C. Appeared.

2.7 Formulations suitable for stabilization independent of drying method

14 shows that only formulations containing sorbitol can resist stress caused by various drying methods. It can also be further improved by the inclusion of MSG and MgCl ₂ (especially type 3), which forms a formulation that provides a significant or higher recovery with vacuum drying with trehalose or sucrose. From initial results (Experiment E), it was concluded that formulations based on sorbitol, MSG and MgCl ₂ had the best recovery after accelerated stability testing.

2.8 Peptone-free formulation

One of the better formulations without peptone added by the inventors found in the above experiment is a formulation containing 10% sorbitol + 10% MSG + 5% MgCl ₂ . In a further experiment, it was evaluated which of the major excipients in this formulation affected. The results were compared to two standard formulations based on sucrose and trehalose. The results immediately after freeze drying are shown in FIG. 15. From FIG. 15, it was concluded that mainly sorbitol was responsible for the recovery of IPV immediately after lyophilization, especially for subtypes 1 and 2. The combination of MSG and MgCl ₂ shows a significant stabilization of the 3 IPV subtype. Compared to sorbitol alone, the combination of sorbitol, MSG and MgCl ₂ showed the best recovery after lyophilization, especially for type 3.

When evaluating the accelerated stability as shown in Figure 16, most of the formulations showed a significant reduction in recovery, as well as formulations containing only sorbitol. However, formulations containing 10% sorbitol + 10% MSG + 5% MgCl ₂ (with / without mannitol) showed high recovery after storage at 45 ° C. for 1 week.

references

1. Bonnet, M.C. and A. Dutta, World wide experience with inactivated poliovirus vaccine. Vaccine, 2008. 26 (4978): p. 4983.

2. Minor, P., Vaccine-derived poliovirus (VDPV): Impact on poliomyelitis eradication. Vaccine, 2009. 27 (20): p. 2649-2652.

3. Shahzad, A. and G. Kohler, Inactivated Polio Vaccine (IPV): A strong candidate vaccine for achieving global polio eradication program. Vaccine, 27. 2009: p. 5293-5294.

4. Ehrenfeld, E., J. Modlin, and K. Chumakov, Future of polio vaccines. Expert Rev Vaccines, August 8, 2009: p. 899-905.

5. John, J., Role of injectable and oral polio vaccines in polio eradication. Expert Rev Vaccines, 2009. 8 (1): p. 5-8.

6. Westdijk, J., et al., Characterization and standardization of Sabin based inactivated polio vaccine: proposal for a new antigen unit for inactivated polio vaccines. Vaccine, 2011. 29 (18): p. 3390-7.

7.Clements, C.J., G. Larsen, and L. Jodar, Technologies that make administration of vaccines safer. Vaccine, 22. 2004 (15-16): p. 2054-2058.

8. Friede, M. and M.T. Aguado, Need for new vaccine formulations and potential of particulate antigen and DNA delivery systems. Adv Drug Deliv Rev, 2005. 57 (3): p. 325-331.

9. Moynihan, M. and I. Petersen, The durability of inactivated poliovirus vaccine: studies on the stability of potency in vivo and in vitro. J Biol Stand, 1982. 10 (3): p. 261-8.

10. WHO, Temperature sensitivity of vaccines. 2006.

11.Sawyer, L.A., et al., Deleterious effect of thimerosal on the potency of inactivated poliovirus vaccine. Vaccine, 12 (9) 1994: p. 851-6.

12. Vidor, The place of DTP / eIPV vaccine in routine paediatric vaccination. Rev Med Virol, 1994. 4: p. 261-77.

13. Bruce Aylward, R., et al., Risk management in a polio-free world. Risk Anal, 2006. 26 (6): p. 1441-8.

14. Sutter, R.W., V.M. Caceres, and P. Mas Lago, The role of routine polio immunization in the post-certification era. Bull World Health Organ, 2004. 82 (1): p. 31-9.

15. Thompson, K.M. and R.J. Duintjer Tebbens, The case for cooperation in managing and maintaining the end of poliomyelitis: stockpile needs and coordinated OPV cessation. Medscape J Med, 2008. 10 (8): p. 190.

16. Tebbens, R.J., et al., Optimal vaccine stockpile design for an eradicated disease: application to polio. Vaccine, 2010. 28 (26): p. 4312-27.

17. Amorij, J.P., et al., Development of stable influenza vaccine powder formulations: challenges and possibilities. Pharm Res, 2008. 25 (6): p. 1256-73.

18. Wang, W., Lyophilization and development of solid protein pharmaceuticals. Int J of Pharm, 2000. 203: p. 1-60.

19. Chang, L. and M.J. Pikal, Mechanisms of protein stabilization in the solid state. J of Pharm Sc, 2009. 98 (9): p. 2886-2908.

20. Arakawa, T., et al., Factors affecting short-term and long-term stabilities of proteins. Adv Drug Deliv Rev, 2001. 46 (1-3): p. 307-26.

21.Bhatnagar, B.S., R.H. Bogner, and M.J. Pikal, Protein stability during freezing: separation of stresses and mechanisms of protein stabilization. Pharm Dev Technol, 2007. 12 (5): p. 505-23.

22. Skrabanja, A.T., et al., Lyophilization of biotechnology products. PDA J Pharm Sci Technol, 1994. 48 (6): p. 311-7.

23. Dias, C.L., et al., The hydrophobic effect and its role in cold denaturation. Cryobiology, 2010. 60 (1): p. 91-9.

24.Carpenter, J.F. and J.H. Crowe, The mechanism of cryoprotection of proteins by solutes. Cryobiology, 25 (3), 1988: p. 244-55.

25. Chang, B.S., B.S. Kendrick, and J.F. Carpenter, Surface-induced denaturation of proteins during freezing and its inhibition by surfactants. J Pharm Sci, 1996. 85: p. 1325-1330.

26. Tang, X.C. and M.J. Pikal, Measurement of the kinetics of protein unfolding in viscous systems and implications for protein stability in freeze-drying. Pharm Res, 2005. 22 (7): p. 1176-85.

27.Webb, S.D., et al., A new mechanism for decreasing aggregation of recombinant human interferon-gamma by a surfactant: Slowed dissolution of lyophilized formulations in a solution containing 0.03% polysorbate 20. J Pharm Sci, 2002. 91: p. 543-558.

28.Rupley, J.A. and G. Careri, Protein hydration and function. Adv Protein Chem, 1991. 41: p. 37-172.

29.Carpenter, J.F., T. Arakawa, and J.H. Crowe, Interactions of stabilizing additives with proteins during freeze-thawing and freeze-drying. Dev Biol Stand, 1992. 74: p. 225-38; discussion 238-9.

30. Carpenter, J.F. and J.H. Crowe, An infrared spectroscopic study of the interactions of carbohydrates with dried proteins. Biochemistry, 1989. 28 (9): p. 3916-22.

31.Tian, F., S. Sane, and J.H. Rytting, Calorimetric investigation of protein / amino acid interactions in the solid state. Int J Pharm, 2006. 310: p. 175-186.

32.Akers, M.J., et al., Glycine crystallization during freezing: the effects of salt form, pH, and ionic strength. Pharm Res, 1995. 12 (10): p. 1457-61.

33.Mernn, M., et al., Formulation of proteins in vacuum-dried glasses. II. Process and storage stability in sugar-free amino acid systems. Pharm Dev Technol, 1999. 4 (2): p. 199-208.

34. Costantino, H.R., et al., Deterioration of lyophilized pharmaceutical proteins. Biochemistry (Mosc), 1998. 63 (3): p. 357-63.

35.Kadoya, S., et al., Freeze-drying of proteins with glass-forming oligosaccharide-derived sugar alcohols. Int J Pharm, 2010. 389 (1-2): p. 107-13.

36.Li, S., et al., Effects of reducing sugars on the chemical stability of human relaxin in the lyophilized state. J Pharm Sci, 1996. 85 (8): p. 873-7.

37. Franks, F., Long-term stabilization of biologicals. Biotechnology (N Y), 1994. 12 (3): p. 253-6.

38. Yu, L., et al., Existence of a mannitol hydrate during freeze-drying and practical implications. J Pharm Sci, 1999. 88 (2): p. 196-8.

39.Hancock, B.C. and G. Zografi, Characteristics and significance of the amorphous state in pharmaceutical systems. J Pharm Sci, 1997. 86 (1): p. 1-12.

40. Schmitt, E.A., D. Law, and G.G. Zhang, Nucleation and crystallization kinetics of hydrated amorphous lactose above the glass transition temperature. J Pharm Sci, 1999. 88 (3): p. 291-6.

41. Fox, K.C., Biopreservation. Putting proteins under glass. Science, 1995. 267 (5206): p. 1922-3.

42.Crowe, J.H., J.F. Carpenter, and L.M. Crowe, The role of vitrification in anhydrobiosis. Annu Rev Physiol, 1998. 60: p. 73-103.

43. van Ingen, C. and C.S. Tan, Preservation mixture and use thereof, W.I.P. Organization, Editor. 2010, De Staat Der Nederlanden, vert. door de minister van VWS.

44. Bam, N.B., et al., Tween protects recombinant human growth hormone against agitation-induced damage via hydrophobic interactions. J Pharm Sci, 1998. 87 (12): p. 1554-9.

45. Christensen, D., et al., Trehalose preserves DDA / TDB liposomes and their adjuvant effect during freeze-drying. Biochim Biophys Acta, 2007. 1768 (9): p. 2120-2129.

46.Izutsu, K., S. Yoshioka, and T. Terao, Effect of mannitol crystallinity on the stabilization of enzymes during freeze-drying. Chem Pharm Bull (Tokyo), 42/1/1994: p. 5-8.

47.Kim, A.I., M.J. Akers, and S.L. Nail, The physical state of mannitol after freeze-drying: effects of mannitol concentration, freezing rate, and a noncrystallizing cosolute. J Pharm Sci, 1998. 87 (8): p. 931-5.

48.Kreilgaard, L., et al., Effects of additives on the stability of recombinant human factor XIII during freeze-drying and storage in the dried solid. Arch Biochem Biophys, 1998. 360 (1): p. 121-34.

49. Leal, M.L., et al., Study on thermostabilizers for trivalent oral poliomyelitis vaccine. Mem Inst Oswaldo Cruz, 1990. 85 (3): p. 329-38.

50.Liska, V., et al., Evaluation of a recombinant human gelatin as a substitute for a hydrolyzed porcine gelatin in a refrigerator-stable Oka / Merck live varicella vaccine. J Immune Based Ther Vaccines, May 5, 2007: p. 4.

51. Melnick, J.L., Thermostability of poliovirus and measles vaccines. Dev Biol Stand, 1996. 87: p. 155-60.

52. Nagel, J., et al., Some experiments on freezedrying of inactivated poliomyelitis-vaccines. Arch Gesamte Virusforsch, 12 December 1963: p. 718-20.

53.Ohtake, S., et al., Heat-stable measles vaccine produced by spray drying. Vaccine, 2010. 28 (5): p. 1275-84.

54. Sood, D.K., et al., Study on the stability of 17D-204 yellow fever vaccine before and after stabilization. Vaccine, 11 (11) 1993: p. 1124-8.

55. Ungar, J., et al., Preparation and properties of a freeze-dried B.C.G. vaccine of increased stability. Br Med J, 1962. 2 (5312): p. 1086-9.

56. Wang, W., Instability, stabilization, and formulation of liquid protein pharmaceuticals. Int J Pharm, 1999. 185 (2): p. 129-88.

57. Wright, D., P.W. Muggleton, and M.I. Griffiths, Evaluation of the stability of dried BCG vaccine. Tubercle, 1972. 53 (2): p. 92-9.

58. Izutsu, K., C. Yomota, and N. Aoyagi, Inhibition of mannitol crystallization in frozen solutions by sodium phosphates and citrates. Chem Pharm Bull (Tokyo), 2007. 55 (4): p. 565-70.

59.Shiomi, H., T. Urasawa, and S. Urasawa, Heat stability of the lyophilized Sabin poliovaccine. Jpn J Infect Dis, 2003. 56 (2): p. 70-2.

60. Burke, C.J., et al., The adsorption of proteins to pharmaceutical container surfaces. International Journal of Pharmaceutics, 1992. 86 (1): p. 89-93.

61.Kersten, G., T. Hazendonk, and C. Beuvery, Antigenic and immunogenic properties of inactivated polio vaccine made from Sabin strains. Vaccine, 17. 1999 (15-16): p. 2059-2066.

62.Doel, T.R., et al., The evaluation of a physical method for the quantification of inactivated poliovirus particles and its relationship to D-antigenicity and potency testing in rats. J Biol Stand, 1984. 12 (1): p. 93-9.

63. Ivanov, A.P. and E.M. Dragunsky, ELISA as a possible alternative to the neutralization test for evaluating the immune response to poliovirus vaccines. Expert Rev Vaccines, 2005. 4 (2): p. 167-72.

64. van Steenis, G., A.L. van Wezel, and V.M. Sekhuis, Potency testing of killed polio vaccine in rats. Dev Biol Stand, 1981. 47: p. 119-28.

65. Wood, DJ and AB Heath, A WHO collaborative study of immunogenicity assays of inactivated poliovirus vaccines. Biologicals, 23.4.1995: p. 301-11.

Abbreviation

BCG-Bacillus Calmette-Guerin

DoE-Design of Experiments

DSC-Differential Scanning Calorimetry

DU / D-Ag-D-Unit / D-antigenicity

ELISA-Enzyme-Linked ImmunoSorbent Assay

HES-Hydroxyethyl starch

HRP-Horseradish peroxidase

IgG-Immunoglobulin G

IPV-Inactivated Polio Vaccine

MAN-Mannitol

MS-Mass Spectrometry

MSG-Mono-Sodium Glutamate

OPV-Oral Polio Vaccine

PEP-Peptone

QC-Quality Control (department at RIVM)

RIVM-National Institute for Public Health and Environment

RMC-Residual Moisture Content

RP-HPLC-Reversed-Phase High-Performance Liquid Chromatography

sIPV-Sabin Inactivated Polio Vaccine (based on Sabin strains)

SOR-Sorbitol

SUC-Sucrose

T _g -Glass transition temperature

TREH-Trehalose

VAPP-Vaccine Associated Paralytic Poliomyelitis

VDPV-Vaccine Derived Poliovirus

WHO-World Health Organization

Claims (23)

Drying the solution containing biopharmaceuticals, amino acids, polyols, metal salts and water,
The amino acid is glutamate, arginine, histidine, asparagine, lysine, leucine, glycine or mixtures thereof,
The solution comprises at least 2.0% (w / v) polyol, the polyol being a sugar or sugar alcohol, and
The solution comprises at least 0.2% (w / v) of a metal salt, wherein the metal salt is a divalent cation, a metal salt of Li ⁺ , or mixtures thereof.
According to claim 1,
The solution consists essentially of 1 pg-10 g of biopharmaceutical per ml, 0.01-20% (w / v) amino acid, 2.0-20% (w / v) polyol, 0.2-10% (w / v) metal salt and A method of making a biopharmaceutical in a dry formulation, consisting of water.
According to claim 1,
a) the polyol is sorbitol, mannitol, mannose, maltitol or mixtures thereof;
b) The metal salt is a salt of Mg ⁺⁺ , Ca ⁺⁺ or Li ⁺ or a mixture thereof,
Method for preparing a biopharmaceutical in a dried formulation.
According to claim 1,
The metal salt is Cl ^- or SO ₄ ^2- salt or mixtures thereof,
Method for preparing a biopharmaceutical in a dried formulation.
According to claim 3,
The glutamate is dissolved in solution in the form of monosodium glutamate, the arginine is present in the form of poly-L-arginine, and the lysine is present in the form of poly-L-lysine,
Method for preparing a biopharmaceutical in a dried formulation.
The method of claim 5,
The solution consists essentially of 1 pg to 10 g of biopharmaceutical per ml, 5-20% (w / v) sorbitol, 5-20% (w / v) monosodium glutamate, and 2-10% (w / v) Consisting of magnesium salts of,
Method for preparing a biopharmaceutical in a dried formulation.
The method of claim 6,
The magnesium salt is MgCl ₂ , MgSO ₄ or a mixture thereof,
Method for preparing a biopharmaceutical in a dried formulation.
The method of claim 6,
The solution further comprises 5-20% (w / v) mannitol,
Method for preparing a biopharmaceutical in a dried formulation.
The method according to any one of claims 1 to 8,
The solution contains a pharmaceutically acceptable buffer, buffered at neutral pH,
Method for preparing a biopharmaceutical in a dried formulation.
The method according to any one of claims 1 to 8,
The solution is dried by air drying, vacuum drying, spray drying or freeze drying,
Method for preparing a biopharmaceutical in a dried formulation.
The method according to any one of claims 1 to 8,
The biologic is a preparation comprising a protein, a polypeptide or a peptide,
Method for preparing a biopharmaceutical in a dried formulation.
The method according to any one of claims 1 to 8,
The biologic is a virus,
Method for preparing a biopharmaceutical in a dried formulation.
The method according to any one of claims 1 to 8,
The biological agent is Enterovirus ( Enterovirus ) or pneumovirus ( Pneumovirus ),
Method for preparing a biopharmaceutical in a dried formulation.
The method of claim 11,
The biologic includes serotype 1, 2 and 3 poliovirus,
Method for preparing a biopharmaceutical in a dried formulation.
The method of claim 11,
The biopharmaceutical comprises inactivated poliovirus of serotypes 1, 2 and 3,
Method for preparing a biopharmaceutical in a dried formulation.
The method of claim 11,
Wherein the biologic is human or bovine RSV,
Method for preparing a biopharmaceutical in a dried formulation.
The method of claim 11,
The biologic is RSV comprising a viral genome with a deleted or inactivated G adhesion protein gene,
Method for preparing a biopharmaceutical in a dried formulation.
The method according to any one of claims 1 to 8,
The dried formulation, upon reconstitution after drying, retains at least 50% of the activity of the biopharmaceutical present in the solution prior to drying,
Method for preparing a biopharmaceutical in a dried formulation.
The method of claim 18,
The solution to be dried contains more than one other biopharmaceutical, wherein the difference in activity loss for other biopharmaceuticals is less than 50%, so the retention activity of the biopharmaceutical with the highest loss of activity is the lowest set at 100% Expressed as a percentage of the retention activity of the biologic with loss,
Method for preparing a biopharmaceutical in a dried formulation.
The method according to any one of claims 1 to 8,
The dried formulation retains at least 50% of the activity of the biopharmaceutical present in the solution prior to drying, upon reconstitution after storage for at least one week at 45 ° C
Method for preparing a biopharmaceutical in a dried formulation.
The method of claim 20,
The dried formulation comprises more than one other biologic, wherein the difference in activity loss for the other agent is less than 50%, so the retention activity of the biologic with the highest loss of activity is the lowest set at 100% Expressed as a percentage of the retention activity of the biologic with loss,
Method for preparing a biopharmaceutical in a dried formulation.
As a formulation of a protein, polypeptide or peptide, the formulation further comprises amino acids, polyols, and metal salts and water,
The amino acid is glutamate, arginine, histidine, asparagine, lysine, leucine, glycine or mixtures thereof,
The formulation comprises at least 2.0% (w / v) polyol, the polyol being a sugar or sugar alcohol, and
The formulation comprises at least 0.2% (w / v) of a metal salt, wherein the metal salt is a divalent cation, a metal salt of Li ⁺ , or a mixture thereof, a formulation used for the prevention or treatment of polio. delete

Images